title: Transmembrane domain of cystic fibrosis transmembrane conductance regulator: design, characterization, and secondary structure of synthetic peptides m1-m6.
abstract: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) give rise to cystic fibrosis (CF), the most common genetic disease in the Caucasian population. CFTR is organized into five putative domains, including two that are predicted to be transmembrane and consist of six membrane-spanning segments each. CFTR mediates regulated anion transport across the apical membrane of epithelial cells. The pore through which CFTR transports its solutes is thought to be formed by some combination of the amino-terminal membrane-spanning segments. Although these sequences are predicted to be alpha-helical in secondary structure, to date, no direct structural evidence has been presented testing this hypothesis. Here, we present the biophysical characterization of six peptides (m1-m6) representing the predicted amino-terminal membrane-spanning domain of CFTR. The peptides can be incorporated into liposomes and are soluble in SDS micelles and trifluoroethanol (TFE). FTIR and CD spectroscopy indicate all six peptides adopt a stable, predominantly alpha-helical secondary structure in these environments. In contrast, peptide m6 undergoes a shift from alpha-helix to beta-sheet when dissolved in 20% methanol. Additionally, the peptides show an increase in beta-sheet in TFE, a known inducer of alpha-helices, relative to that seen in the nativelike environments. These results have implications for the folding of this complex membrane protein and suggest that the possible functional role of m6 is manifested through a shift in secondary structure.
authors.affiliation: Department of Physiology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas 75235-9040, USA.
authors.forename: W C
authors.initials: WC
authors.lastname: Wigley
authors.forename: S
authors.initials: S
authors.lastname: Vijayakumar
authors.forename: J D
authors.initials: JD
authors.lastname: Jones
authors.forename: C
authors.initials: C
authors.lastname: Slaughter
authors.forename: P J
authors.initials: PJ
authors.lastname: Thomas
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000138:chemical synthesis* / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D000432:Methanol; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D008565:Membrane Proteins; D010455:Peptides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000432:Methanol